The ubiquitin-proteasome pathway is a major cellular proteolysis machinery that selectively targets cellular proteins for degradation. Protein knockout, a new technology we recently developed, harnesses the specificity of the ubiquitin proteolytic system to direct the degradation of otherwise stable cellular proteins. The long-range goal of this research plan is to apply the protein knockout system as a rapid molecular analysis tool to decipher the function of cellular oncoproteins and to validate their potential use as drug targets. The objective of this application is to improve the efficiency and specificity of the protein knockout system and to assess its efficacy in the proteolytic removal of the overexpressed c-myc oncoprotein in established cell culture and animal models for leukemogenesis. The rationale is that recognition of specific cellular proteins by the engineered substrate receptors of the ubiquitination machinery allows for their degradation by the ubiquitin-proteasome pathway. The research plan has been formulated on the basis of strong preliminary data, and on recent studies by many laboratories including our own. We are uniquely prepared to undertake the proposed research, because we have strong preliminary data demonstrating the efficacy of the protein knockout technology in the selective degradation of stable cellular proteins. The objective of the application will be accomplished by pursuing the following specific aims: (1) R21 phase: To improve the efficiency, specificity and delivery of the protein knockout system. (2) R33 phase: To evaluate targeted c-myc degradation in the inhibition of oncogenic transformation in established cell culture systems. (3) R33 phase: To antagonize myc-mediated tumorigenecity by protein knockout in mouse models for leukemogenesis. The proposed work is innovative, because it capitalizes on the recently identified properties of the ubiquitination machinery by our group and by others. It is our expectation that this approach will offer an efficient means to downregulate the c-myc oncoprotein at the protein level and to inhibit c-myc-mediated neoplastic transformation. These results are significant, because they are expected to provide a comprehensive evaluation of the protein knockout technology as a simple and cost effective molecular analysis tool to elucidate the function of cancer-related proteins and genetic pathways, and to validate their potential use as targets for therapeutic intervention.